Respiratory Syncytial Virus G Protein Research Articles

Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

Obtaining and Characterization of the Monoclonal Antibodies Against G-Protein of the Respiratory Syncytial Virus

The aim of this study was to obtain hybridomas producing monoclonal antibodies (Mabs) to the G-protein of the respiratory syncytial virus (RSV), and to evaluate their immunological characteristics and virus-neutralizing activity.Material and methods. Mouse Mabs were obtained using hybridoma technology. The properties of Mabs were studied by enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining (IF) of infected cells, as well as by biological neutralization test in vitro (NT). To identify epitopes recognized by the Mabs on G protein ELISA additivity test was used.Results. Hybridization of splenocytes with Sp2/0 myeloma cells and primary screening showed that 75 hybridomas produce antibodies interacting with purified virus, 17 of them also react with the recombinant G-protein in ELISA. In NT 4, hybridomas suppressed in vitro RSV infection by more than 50%. Cloning of these hybridomas revealed 4 monoclones producing the most active Mabs. Mab 1C11 was IgG2a, 3 others (5D4, 5G11 and 6H4) were IgM. Three IgM Mabs actively reacted with both RSV A2 and Long, and with G-protein; Mab 1C11 was less reactive with all antigens tested. All Mabs suppressed RSV infection, while Mab 5D4 supressed it almost completely (98%). IF analysis showed that all Mabs detected RSV G-protein in the cell cytoplasm, the largest number of infected cells was detected using Mab 5D4 (80%). It was shown that the isolated Mabs were directed to two non-overlapping epitopes on the RSV G-protein.Conclusion. The isolated Mabs can be used to detect RSV in clinical samples by ELISA and IF. The isolated Mabs can be used for humanized recombinant antibodies construction and for the treatment of RSV infection in future.

High epidemic burden of RSV disease coinciding with genetic alterations causing amino acid substitutions in the RSV G-protein during the 2016/2017 season in The Netherlands

BackgroundWe found amino acid substitutions in the Gglycoprotein of respiratory syncytial virus (RSV) A during the 2016/2017 epidemic in The Netherlands. ObjectivesWe evaluated whether these alterations led to increased RSV incidence and disease burden. Study designWe sequenced the gene encoding the G-protein of prospectively collected clinical specimens from secondary care adult patients testing positive for RSV during the 2016/2017 and 2017/2018 epidemic RSV season. We evaluated associations between genetic, clinical and epidemiological data. ResultsWe included 49 RSV strains. In 2016/2017 28 strains were included, 20 community acquired RSV-A, 5 hospital acquired RSV-A and 3 community acquired RSV-B. In 2017/2018 21 strains were included, 8 community acquired RSV-A and 13 community acquired RSV-B. G-proteins of 10 out of the 20 community acquired 2016/2017 RSV-A strains shared a set of eight novel amino acid substitutions of which seven in mucin-like regions 1 and 2 and one in the heparin binding domain. This genetic variant was no longer detected among 2017/2018 RSV-A strains. Among patients carrying the novel RSV-A strain-type, 30% died. ConclusionsA set of eight amino acid substitutions was found in 50% of the 2016/2017 community acquired RSV-A G-proteins. This combination of substitutions was globally never observed before. The appearance of this new strain-type coincided with an increased RSV peak in The Netherlands and was associated with higher disease severity. The transient character of this epidemic strain-type suggests rapid clearance of this lineage in our study community.

Replacement and Positive Evolution of Subtype A and B Respiratory Syncytial Virus G-Protein Genotypes From 1997–2012 in South Africa

Of the respiratory syncytial virus (RSV) genotypes previously described in South Africa during 1997-2002, only GA2 and GA5 persisted until 2006, with BA having replaced all previous RSV-B genotypes. This poses the question whether RSV-A is more stable than RSV-B and whether positive selection drives evolution of genotypes. RSV-positive specimens were randomly selected during 2009-2012, subtyped, sequenced, and compared to RSV recovered from specimens obtained during 1997-2001 and 2006-2009. Bayesian phylogenetic analysis was performed on the G-protein. Phylogenetic analysis indicated that RSV-A genotype GA2 dissolved to form SAA2 (unique to South Africa), NA1 and NA2 (identified in Japan), and ON1 (identified in Canada and having a 72-bp insertion) and that GA5 drifted from 1999-2012 to form 3 subgenotypes (GA5 I-III). RSV-B genotypes all had the 60-bp insertion typical of BA genotypes but clustered into subgenotypes BA8-10. Positive selection was identified in the G-protein of both subtypes, but RSV-A's rate of evolution was slower than that of RSV-B, with the most recent common ancestors dating from 1945 and 1951, respectively. Seven new positively selected sites were identified in South African strains, 2 for RSV-A and 5 for RSV-B. Positive selection drove both RSV-A and -B genotypes to evolve, resulting in replacement of all genotypes over the 15-year study period in South Africa.

Therapeutic targeting of respiratory syncytial virus G-protein

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in infants and young children and an important pathogen of the elderly and immune suppressed. The only intervention currently available is a monoclonal antibody against the RSV fusion protein, which has shown utility as a prophylactic for high-risk premature infants, but which has not shown postinfection therapeutic efficacy in the specific RSV-infected populations studied. Thus, for the major susceptible populations, there remains a great need for effective treatment. Recent results support monoclonal antibody targeting of the RSV G-protein for therapeutic use. This objective encompasses a dual mechanism: reduction in the ability of RSV G-protein to distort the host innate immune response, and direct complement-mediated antiviral activity.

Molecular epidemiology of respiratory syncytial virus in children with acute respiratory tract infections and sequence analysis of G gene in Chongqing area

Objective To investigate the epidemiological features of respiratory syncytial virus (RSV) A and B in hospitalized children with acute respiratory tract infections(ARTIs) and to analyze the genetic characteristics of G protein gene of RSV in Chongqing area, especially for BA strains. Methods Nasopharyngeal secretions collected from 508 hospitalized children with ARTls from April, 2008 to March, 2009 were screened for RSV using RT-PCR. Full length G protein gene was amplified by RT-PCR from 10 RSV subtype A and 29 RSV subtype B strains. Results Out of the total 508 specimens, 126 (24.8%) were revealed positive for RSV. RSV subtype A and B viruses accounted for 34.1% and 63.5% of the total positive specimens, respectively. The remaining 2.4% of the specimens were positive for both subtype A and B. At the nucleotide level, identities between the 10 subtype A virus G genes and that of the prototype strain A2 were 91.4% -92.0%, indicating genotype GA2. Identities of the 29 subtype B virus G genes and that of the CH18537 strain were 92.0%-93.0%. Nineteen out of 29 RSV subtype B isolates contained highly repeated 60 nucleotides insertion in G protein gene, namely BA strain. As compared to the prototypes, the RSV G protein gene included nucleotide deletion, insertion, substitutions, especially in the carboxy-terminal third of the G gene. Condnsion RSV has been the major cause of acute respiratory tract infections in children in Chongqing area. Subtype B strains, especially BA strains, were predominant during the study peried. Whether the predominated circulation of BA strain is resulted from enhanced attachment function of G protein remains unknown. Key words: Respiratory syncytial virus(RSV) ; Acute respiratory tract infections; G protein; Nucleotide; Amino acids

Synthesis, refolding and protective immune responses of a potential antigen for human Respiratory Syncytial Virus vaccine.

The design of new antigens with both high immunogenic and safety properties is of particular interest to vaccine against infectious diseases. In the present study, we describe the synthesis and the refolding of peptide G20 derived from the Human Respiratory Syncytial Virus (hRSV) G-protein. G20 (MEF G140-190 G144-158) is a peptide of 69 amino acids with two disulfide bridges, which comprises multiple protective B-cell epitopes. It was deleted of the T helper cell epitope 184-198 of the RSV G-protein, which was found to induce pulmonary pathology after RSV challenge in mice. Interestingly, we showed in the present study that G20 generated a highly protective antibody response against RSV challenge in Balb/c mice. Therefore, G20 represents a new potential antigen for an RSV vaccine.

Isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells

Respiratory syncytial virus (RSV) infection causes severe lower respiratory diseases in infancy, early childhood and the elderly. RSV infections respond poorly to current therapies. Therefore, we initiated a search for novel drug targets by investigating the characteristics and identity of RSV adhesion receptors on mammalian cells. Soluble human lectins, complex polysaccharides and a low molecular selectin antagonist, TBC1269, were used to characterise and isolate the RSV receptor on a human epithelial cell line (Hep2 cells). The binding characteristics of the RSV receptor on Hep2 cells were similar to those reported for L-selectin. The carbohydrate-based selectin antagonists, fucoidan and TBC 1269, inhibit RSV infection both in vitro and in a mouse model of infection. Furthermore, we have isolated annexin II as a potential RSV receptor on Hep2 cells. The expression of annexin II was increased after RSV infection. Recombinant annexin II binds to RSV G-protein, heparin and plasminogen and the binding is inhibited by a selectin antagonist, TBC1269. These findings indicate that inhibitors of annexin II could have potential in treating RSV infection.

Mammalian cell production of a respiratory syncytial virus (RSV) candidate vaccine recovered using a product-specific affinity column.

The recombinant production of a respiratory syncytial virus (RSV) candidate vaccine BBG2Na in baby hamster kidney cells (BHK-21 cells) was investigated. BBG2Na consists of a serum-albumin-binding region (BB) fused to a 101-amino-acid fragment of the RSV G-protein. Semliki Forest virus-based expression vectors encoding both intracellular and secreted forms of BBG2Na were constructed and found to be functional. Affinity recovery of BBG2Na employing human serum albumin columns was found to be inefficient due to the abundance of BSA in the applied samples. Instead, a strategy using a tailor-made affinity ligand based on a combinatorially engineered Staphylococcus aureus protein A domain, showing specific binding to the G-protein part of the product, was evaluated. In conclusion, a strategy for production and successful recovery of BBG2Na in mammalian cells was created, through the development of a product-specific affinity column.

Human respiratory syncytial virus vaccine antigen produced in plants.

Human respiratory syncytial virus (RSV) is the primary cause of respiratory infection in infants worldwide. Currently there is no available vaccine, although studies in animal models have demonstrated protective immunity induced by an epitope of the RSV G-protein representing amino acids 174-187. Two peptides containing amino acids 174-187 of the G-protein of the human RSV A2 strain (NF1-RSV/172-187 and NF2-RSV/170-191) were separately engineered as translational fusions with the alfalfa mosaic virus coat protein and individually expressed in Nicotiana tabacum cv. Samsun NN plants through virus infection. RSV G-protein peptides were expressed in infected plant tissues at significant levels within 2 wk of inoculation and purified as part of recombinant alfalfa mosaic virions. BALB/c mice immunized intraperitoneally with three doses of the purified recombinant viruses showed high levels of serum antibody specific for RSV G-protein and were protected against infection with RSV Long strain.

Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells.

Lung surfactant protein A (SP-A) has a central role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-A. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G-protein from RSV (human, A2 strain) and human SP-A. The effect of SP-A on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and by interleukin-8 (IL-8) release from buffy coat (BC) cells. SP-A binds to RSV G-protein in a concentration-dependent manner that is inhibitable by both ethylenediamine tetraacetic acid (EDTA) and mannan, indicating that binding is through the carbohydrate recognition domain of the SP-A and a carbohydrate moiety of the G-protein. The level of RSV infection of Hep-2C cells increases with increasing concentrations of SP-A. The amount of IL-8 released by BC cells in the presence of RSV is increased with SP-A concentrations of 2.9 microg/mL or greater. Our results show that SP-A enhances the attachment of RSV and subsequent entry into host cells. The effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV.

Respiratory syncytial virus G-protein modulates cytokine release from human peripheral blood mononuclear cells

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children and adults. In vivo the host has to cope with intact replicative virus, with non-replicative virus, and/or with viral structural proteins including the outer membrane G-protein. We analyzed the role of purified RSV G-protein with regard to its modulatory efficacy for interleukin (IL) -10, IL-12, and tumor necrosis factor-alpha (TNF-alpha) release from human peripheral blood mononuclear cells (PBMC). These cytokines seem to contribute to the deleterious effect in viral infections. Treatment of PBMC with RSV at a multiplicity of infection of 10 down to 0.001 induced the release of TNF-alpha, IL-10, and IL-12; also time kinetics and dose-responses differed markedly. Stimulation of PBMC with purified RSV G-protein (from 0.001 up to 10 microgram/10(6) PBMC) led only to a pronounced increase in IL-10 within a concentration range from 0.01 up to 0.5 microgram/10(6) PBMC with a maximum between 12 and 18 h of incubation. AT later time points (24, 48, and 72 h) G-protein concentrations above 1 microgram/10(6) PBMC suppressed IL-10, TNF-alpha, and IL-12 release from human PBMC. Coating of PBMC with RSV G-protein suppressed IL-10, TNF-alpha, and IL-12 release after subsequent stimulation with RSV. Our data indicate a regulatory role of RSV G-protein immune responses toward viral infection.